U.S. patent application number 11/885276 was filed with the patent office on 2008-06-05 for method for determining the effectiveness of a treatment for preeclampsia.
Invention is credited to Hamutal Meiri.
Application Number | 20080131867 11/885276 |
Document ID | / |
Family ID | 37989106 |
Filed Date | 2008-06-05 |
United States Patent
Application |
20080131867 |
Kind Code |
A1 |
Meiri; Hamutal |
June 5, 2008 |
Method for Determining the Effectiveness of a Treatment for
Preeclampsia
Abstract
A method for determining the effectiveness of a treatment for
preeclampsia of a pregnant woman at risk for preeclampsia, the
method comprising: (a) determining a first concentration of
placental protein 13 (PP13) in a bodily substance of the woman
obtained prior to the treatment; (b) determining a second
concentration of PP13 in a bodily substance of the woman obtained
after initiation of the treatment; and (c) comparing the first and
second concentrations to a corresponding normal level of PP13 and,
based on the comparison, determining the effectiveness of the
treatment. Diagnostic kits for practicing the method are also
disclosed.
Inventors: |
Meiri; Hamutal; (Tel Aviv,
IL) |
Correspondence
Address: |
NATH & ASSOCIATES
112 South West Street
Alexandria
VA
22314
US
|
Family ID: |
37989106 |
Appl. No.: |
11/885276 |
Filed: |
February 1, 2007 |
PCT Filed: |
February 1, 2007 |
PCT NO: |
PCT/IL07/00131 |
371 Date: |
August 29, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60764344 |
Feb 2, 2006 |
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Current U.S.
Class: |
435/1.3 ; 435/29;
436/86 |
Current CPC
Class: |
G01N 2800/368 20130101;
G01N 33/689 20130101; G01N 2800/52 20130101; G01N 2333/471
20130101 |
Class at
Publication: |
435/1.3 ; 436/86;
435/29 |
International
Class: |
A01N 1/02 20060101
A01N001/02; G01N 33/68 20060101 G01N033/68; G01N 33/50 20060101
G01N033/50 |
Claims
1. A method for determining the effectiveness of a treatment for
preeclampsia of a pregnant woman at risk for preeclampsia,
comprising: (a) determining a first concentration of placental
protein 13 (PP13) in a bodily substance of the woman obtained prior
to the treatment; (b) determining a second concentration of PP13 in
a bodily substance of the woman obtained after initiation of the
treatment; and (c) comparing said first and second concentrations
to a corresponding normal level of PP13 and, based on said
comparison, determining the effectiveness of the treatment.
2. The method of claim 1, wherein said first concentration of PP13
is selected from the group consisting of: (a) a predetermined range
of median PP13 concentrations for said bodily substance in a
plurality of untreated pregnant women at a similar risk for
preeclampsia; or (b) a measured PP13 concentration of the bodily
substance of said pregnant woman prior to receiving the
treatment.
3. The method of claim 1 wherein said bodily substance is selected
from the group consisting of serum, amniotic fluid, urine, saliva,
placental tissue and standardized placenta villi.
4. The method of claim 1, wherein the comparison is made between
single measurements of said first concentration and said second
concentration.
5. The method of claim 1, wherein the comparison is made between a
first slope calculated from a plurality of said first
concentrations measured at two or more succeeding time points
during the pregnancy of the woman and a second slope calculated
from a plurality of said second concentrations measured at two or
more succeeding time points during the pregnancy of the woman.
6. The method of claim 1 for determining the relative effectiveness
of two or more different treatments for preeclampsia, the method
comprising: (a) determining a first concentration of PP13 in a
placental tissue explant of the woman obtained prior to the
treatment; (b) contacting the explant with a first treatment; (c)
determining a second concentration of PP13 in the explant after the
treatment; (d) comparing said first and second concentrations to a
corresponding normal level of PP13 and, based on said comparison,
determining the effectiveness of the first treatment; (e) repeating
steps (a) to (d) with one or more additional treatments; and (f)
comparing the relative effectiveness of the two or more different
treatments.
7. The method of claim 6 wherein the second concentration is
measured within 1-4 days after the placenta explant is contacted
with the treatment.
8. The method of claim 1 wherein in step (d), the effectiveness of
the treatment is determined as follows: (a) if there is no
significant difference between the first and second concentrations,
the treatment is not effective; (b) if the difference between the
second concentration and the normal level of PP13 is significantly
less than the difference between the first concentration and the
normal level, the treatment is effective; (c) if the difference
between the second concentration and the normal level of PP13 is
significantly greater than the difference between the first
concentration and the normal level, the treatment is damaging.
9. The method of claim 8, wherein the plurality of each of the
concentrations is compared to a corresponding plurality of normal
levels of PP13.
10. The method of claim 8 wherein the plurality of concentrations
is determined over a period of 2-3 weeks.
11. The method of claim 1, wherein the comparison is based on the
multiple of the medians (MoM), slope and the woman's likelihood
ratio (LR) with receiver operating characteristic (ROC) curves used
to establish cutoffs for sensitivity and specificity.
12. The method of claim 1, wherein the comparison is based on
normal and diseased populations of pregnant women being divided
into quartiles, and the concentrations of PP13 being classified in
the appropriate quartile.
13. A method for determining the relative effectiveness of two or
more different treatments for preeclampsia of a pregnant woman at
risk for preeclampsia, comprising: (a) providing a plurality of
placental tissue explants standardized for release of PP13 in
response to various preeclampsia treatments; (b) contacting a
bodily substance of the woman with a first placental tissue
explant; (c) contacting said explant with a first treatment and
determining the concentration of PP13 of the explant after said
first treatment; (d) repeating steps (b) and (c) with one or more
additional explants and treatments; and (e) determining the
difference between the concentrations of PP13 after said treatments
and a corresponding normal level of PP13, the treatment resulting
in the smallest difference being the most effective.
14. The method of claim 13 wherein the standardized placenta
explants are cryo-preserved.
15. A diagnostic kit for carrying out the method of claim 13
comprising: (a) a set of anti-preeclampsia drugs; and (b) a set of
standardized placenta explants.
16. A kit according to claim 15 further comprising computer
software providing a calculation model to determine the
effectiveness of the drugs based on the measured PP13 values.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a method for tailoring medications
to treat or prevent preeclampsia, and for monitoring their
effectiveness.
BACKGROUND OF THE INVENTION
[0002] The pregnancy disorder known as preeclampsia (PE) is a
complication of pregnancy occurring in 5-7% of all pregnant women
and it is the second most frequent cause of maternal death during
pregnancy (18% of maternal mortality associated with pregnancy in
the United States). Preeclampsia is defined as a new onset
hypertension developed after 20 weeks of gestation in previously
normotensive women. The World Congress of Hypertension in Pregnancy
has provided the following definition for diagnosing preeclampsia:
a new onset hypertension developed after 20 weeks of gestation of
.gtoreq.90/140 mm Hg (systolic/diastolic, at least one) measured on
two occasions, 4-6 hours apart (and in some cases 4-72 hours
apart), coupled to the appearance of protein in the urine
corresponding to 300 mg/DL in 24 hours collection or 2+ by dipstick
measurement, in women who previously had traces or no protein in
the urine. Severe preeclampsia is defined as preeclampsia in which
the hypertension has reached 160/110 mm Hg (systolic/diastolic, at
least one) coupled to proteinuria of .gtoreq.3+ in dipstick or
>3 gr/dl in 24 hr. Eclampsia is an emergency situation in which
severe preeclampsia is exacerbated into convulsion, stroke and coma
that endanger the life of the mother. To avoid such an emergency
situation, the woman is delivered to remove the baby and the
placenta which cause these effects. HELLP is a severe form of
preeclampsia where the major side effects are hemolysis, elevated
liver enzyme and low platelets.
[0003] Although the proportion of preeclampsia is higher in
developing countries, numbers in the U.S. remain high (5-7%). Fifty
percent of all PE pregnancies are delivered via Cesarean section as
compared with only 15-18% of pregnancies in the entire population.
Recovery from a Cesarean section delivery lengthens the recovery
time. Not only is the procedure more complicated, but it is also
more expensive than vaginal delivery. Women who experience PE
disorders during pregnancy have a 9 times higher risk of
consequently developing cardiovascular diseases and their life
expectancy is significantly lower.
[0004] Early-preeclampsia is a severe form of preeclampsia which
develops early and necessitates delivery before 37 weeks of
gestation (before term). Severe and Early-onset PE are major
hazards for both mother and fetus. According to the NICHD, early PE
accounts for 20-25% of all cases of PE, which means that 1-2 out of
100 pregnant women will be affected by this complication, and the
baby is delivered extremely early, after experiencing a stressful
pre-delivery period. The earlier the delivery occurs, the more
severe are the complications of the baby, due to its low birth
weight and incomplete internal organ maturation, the complications
including blindness, motor and cognitive disorders and life-long
medical disabilities. Babies born prematurely due to preeclampsia
are at increased risk to later develop hypertension, cardiovascular
diseases and diabetes. The early preeclampsia cases delivered
before 34 gestational weeks (GW) are the most severe ones,
responsible for most mortality cases, and the babies born, if they
survive, need on average 6-8 weeks in neonatal intensive care
units. According to the NICHD this is the group for whom early
detection is most essential for its life saving capability and
prevention of prematurity. The only current practice to treat
preeclampsia is to deliver the mother, and when such a delivery
takes place prematurely, a variety of impairments of the newborn
baby appear due to lower birth weight, motor and cognitive
disabilities, and, in very severe cases, in-partum or after partum
death. Many studies have been carried out to identify the risk of
developing preeclampsia early.
[0005] The early detection of risk provides two major
advantages:
[0006] 1) It enables to manage the risk by close surveillance of
the woman. Effective increased surveillance for women at high risk
is in compliance with ACOG guidelines, and the increased
surveillance in high-risk cases significantly improves outcome.
Pregnancy Management Programs were shown to save costs due to close
surveillance coupled with education and awareness programs given to
the participating pregnant women. Among benefits are a drop of
births due to early PE (<34 weeks) only 0.6% of all deliveries
compared with the national average of 1.96%, premature delivery
reduction to 0.9% of all deliveries compared with the national
benchmark of 2.3% and low birth weight due to preeclampsia being
1.3% compared with the national average of 2.9%. The close
surveillance allows the pregnant woman to reach a tertiary level
medical center before birth, an issue of great significance in
community clinics and rural-based health service settings. There,
close surveillance enables in certain cases to extend pregnancy
duration so as to reduce the severity of the consequences to the
baby who is under a risk to be delivered pre-maturely. The other
benefit is to buy time to administer treatments and drugs such as
antenatal corticosteroids that facilitate the maturation of fetal
organs.
[0007] 2) The early detection enables a longer period for
developing drug intervention strategies using various putative
agents that are considered to work on the placenta to
prevent/reduce the risk. Although there is no gold standard for
treatment, a number of candidates have shown promise, including low
dose aspirin, low molecular weight heparin, anti oxidants such as
vitamin C and E and magnesium sulfate, among others. In all of
these studies not all women at risk benefited from the therapeutic
intervention. While in some cases there are indications that the
intervention was initiated too late, in other cases there is no
clear evidence to indicate if the medication used wasn't the right
one or wasn't used at the right time or dose. Current studies show
that it is necessary to tailor drug intervention to each woman from
a putative medications list available today (as well as medications
that will become available in due course), and to continuously
monitor the effectiveness of the treatment.
[0008] Among the current leading agents to prevent preeclampsia
are:
[0009] (1) Low dose salicylic acid (aspirin) to improve the blood
flow to the maternal arteries supplying oxygen and nutrients to the
placenta; (2) anti-coagulants such as low molecular weight heparin
were found effective in preventing trombophilia and its
complications that occur in recurrent and severe preeclampsia; (3)
Magnesium Sulfate (MgSO.sub.4) that has so far been proven
effective only for treating eclampsia. However, its usefullness in
treating preeclampsia remains under debate. Nevertheless,
MgSO.sub.4 remains the first-line agent in many institutions for
treating women with preeclampsia and HELLP (hemolysis, elevated
liver enzymes and low platelet count). (4) Anti-oxidants such as
vitamins C and E were shown to reduce the prevalence of
preeclampsia among high-risk pregnancies. In many of these
treatments, side effects such as brain hemorrhages, neuromuscular
blockade and difficult resuscitation can develop and cause
complications to the mother and the fetus.
[0010] Placental Protein 13 (PP13) is a protein of 15-16,000 MW
which may be purified from human placental tissue or prepared by
recombinant technology as described in U.S. Pat. No. 6,548,306
(Admon, et al), the contents of which are incorporated herein by
reference. Purified PP13 was used to develop an assay for the
detection of some pregnancy-related disorders such as intrauterine
growth restriction (IUGR), preeclampsia and preterm delivery as
described in U.S. Pat. No. 5,198,366 (Silberman), the contents of
which are incorporated herein by reference. Both a radioimmunoassay
(RIA) and an enzyme-linked immunosorbent assay (ELISA) were
developed using labeled PP13 and anti PP13 polyclonal
antiserum.
[0011] Amino acid composition and sequence analysis of PP13
revealed highest homology to the galectin family--a group of
proteins with high affinity to sugar residues which is particularly
important in bridging cells to the extracellular matrix (and in
differentiation) (Than, N. G., et al (1999) Placenta 20:703-710;
Than, et al., (2004) Eur. J. Biochem. 271(6):1065-1078). Indeed
PP13 was found by immunohistochemistry to be important in
placentation.
[0012] U.S. Pat. No. 6,790,625, the contents of which are
incorporated herein by reference, discloses monoclonal antibodies
to PP13 and a solid-phase immunoassay capable of measuring maternal
serum PP13 during the early stages of pregnancy.
[0013] WO 04/021012, the contents of which are incorporated herein
by reference, discloses a diagnostic method for pregnancy
complications based on a number of factors, including PP13
level.
SUMMARY OF THE INVENTION
[0014] It is an object of the present invention to provide a simple
in-vitro assay that will allow an attending physician to monitor
the effectiveness of putative anti-preeclampsia medications
administered to a pregnant woman who is at an elevated risk for or
suffering from preeclampsia.
[0015] It is another object of the present invention to use an
in-vitro or ex-vivo system to tailor drug intervention for
preventing or treating preeclampsia from a list of putative
anti-preeclampsia medications.
[0016] In a first aspect of the present invention, there is
provided a method for determining the effectiveness of a treatment
for preeclampsia of a pregnant woman at risk for preeclampsia,
comprising: [0017] (a) determining a first concentration of
placental protein 13 (PP13) in a bodily substance of the woman
obtained prior to the treatment; [0018] (b) determining a second
concentration of PP13 in a bodily substance of the woman obtained
after initiation of the treatment; and [0019] (c) comparing the
first and second concentrations to a corresponding normal level of
PP13 and, based on the comparison, determining the effectiveness of
the treatment.
[0020] This aspect of the invention may be referred to at times as
the direct method.
[0021] Optionally the method of the invention may be continued
until delivery to follow the effectiveness of the treatment.
[0022] In the present specification, the term "preeclampsia" (PE)
includes all types of the disease, including mild, severe, early
onset, late onset, PE complicated by intrauterine growth
restriction (IUGR), and HELLP, unless specifically indicated
otherwise.
[0023] The term "a treatment for preeclampsia" includes all types
of medical treatments used to prevent, reduce the severity of, or
therapeutically treat preeclampsia such as hyperoxia and, in
particular, treatments using drugs or food additives. Non-limiting
examples of drugs for the treatment of preeclampsia include
anti-platelet agents such as low dose aspirin, anti-coagulants such
as heparins including low molecular weight heparin, anti-oxidants
such as vitamins C and E, and magnesium sulfate, as well as novel
experimental treatments with growth factors such as vascular
epidermal growth factors (VEGF), treatment with CO, etc.
[0024] The term "determining the effectiveness of a treatment" may
include both comparing the effectiveness of one type of treatment
to another type, comparing the same type of treatment under
different conditions (oxygen level, temperature, etc), as well as
monitoring the effectiveness of a particular treatment over
time.
[0025] The term "bodily substance of the woman" includes body
fluids (serum, amniotic fluid, urine, saliva) and placenta tissue
obtained by way of chorionic villous sampling (CVS), amniocentesis,
placenta biopsy or using standardized placenta villi.
[0026] The term "normal level of PP13" refers to the level of PP13
found in a bodily substance of a normal, healthy pregnant woman who
has not developed preeclampsia or is not at risk to develop
preeclampsia. It may also refer at times to the level of PP13
released from explants of placenta obtained after delivery, from
cultured placenta cells derived from amniocentesis or from
placental villi isolated after chorionic villi sampling from a
normal, healthy pregnant woman who has not developed preeclampsia
or is not at risk to develop preeclampsia.
[0027] The level of PP13 can vary as a function of time
(gestational weeks), as a function of the genetic and physical
characteristics of the woman such as body mass index, maternal age,
ethnicity, and parity, and as a function of the identity of the
bodily substance measured. Therefore, when comparing a measured
PP13 value from a patient to the normal level of PP13, these
parameters should be taken into account. At times, the measured
PP13 value will be normalized in order to compare it to the
corresponding normal level of PP13.
[0028] A woman at high-risk to develop preeclampsia may be
determined by: 1) risk factors (such as preeclampsia in previous
pregnancy or family history); 2) impaired blood flow to the
maternal uterine arteries as assessed by higher pulsatility index
measured by Doppler ultrasound; 3) abnormal level of various serum
markers such as PP13 in the pregnant woman's body fluids as
disclosed in the aforementioned patents and patent
applications.
[0029] In one embodiment, the first concentration of PP13 is
selected from the group consisting of: (a) a predetermined range of
median PP13 concentrations for the bodily substance in a plurality
of untreated pregnant women at a similar risk for preeclampsia; or
(b) a measured PP13 concentration of the bodily substance of the
pregnant woman prior to receiving the treatment.
[0030] The measurement of changes in PP13 in the bodily substances
in accordance with the method of the invention is used to determine
if the woman's risk persists, is reduced or is elevated in
comparison to her initial risk, and in comparison to the typical
values of PP13 in the bodily substances of a plurality of normal
and high risk women at the respective weeks of gestation. The
continuous redefinition of the woman's risk to develop preeclampsia
is used as a means to assess the effectiveness of putative
medication therapy to reduce/remove the risk to develop
preeclampsia.
[0031] In one embodiment of the invention, the comparison is made
between single measurements of the first concentration and the
second concentration. In another embodiment, the comparison is made
between a first slope calculated from a plurality of the first
concentrations measured at two or more succeeding time points
during the pregnancy of the woman and a second slope calculated
from a plurality of the second concentrations measured at two or
more succeeding time points during the pregnancy of the woman. In a
preferred embodiment, the plurality of concentrations is determined
over a period of 2-3 weeks. In a further embodiment, the plurality
of each of the concentrations is compared to a corresponding
plurality of normal levels of PP13.
[0032] One embodiment of the invention involves placenta tissues.
Assays are carried out in tissue cultured for 1-7 days in tissue
culture medium following a treatment given in-vivo. Another
embodiment of the invention involves exposing the tissue to
putative medications and selecting the most effective one. Although
this saves a woman from exposure to an un-necessary medication, she
is exposed to an interventional procedure of risk. Thus, the use of
placental tissue in the method of the invention is usually suitable
only to those undergoing in any event an interventional sampling of
placenta tissue. Occasionally, women who have repeated history of
preeclampsia and are considered at very high risk for preeclampsia
may be offered this approach as well.
[0033] Thus, the invention also includes a method for determining
the relative effectiveness of two or more different treatments for
preeclampsia, the method comprising: [0034] (a) determining a first
concentration of PP13 in a placental tissue explant of the woman
obtained prior to the treatment; [0035] (b) contacting the explant
with a first treatment; [0036] (c) determining a second
concentration of PP13 in the explant after the treatment; [0037]
(d) comparing the first and second concentrations to a
corresponding normal level of PP13 and, based on the comparison,
determining the effectiveness of the first treatment; [0038] (e)
repeating steps (a) to (d) with one or more additional treatments;
and [0039] (f) comparing the relative effectiveness of the two or
more different treatments.
[0040] In one embodiment, the effectiveness of the treatment is
determined in step (d), as follows: [0041] (a) if there is no
significant difference between the first and second concentrations,
the treatment is determined as ineffective; [0042] (b) if the
difference between the second concentration and the normal level of
PP13 is significantly less than the difference between the first
concentration and the normal level, the treatment is determined as
effective; [0043] (c) if the difference between the second
concentration and the normal level of PP13 is significantly greater
than the difference between the first concentration and the normal
level, the treatment is damaging.
[0044] In a preferred embodiment, the second concentration is
measured within 1-4 days after the placenta explant is contacted
with the treatment
[0045] A second aspect of the invention relates to a method for
determining the relative effectiveness of two or more different
treatments for preeclampsia of a pregnant woman at risk for
preeclampsia, comprising: [0046] (a) providing a plurality of
placental tissue explants standardized for release of PP13 in
response to various preeclampsia treatments; [0047] (b) contacting
a bodily substance of the woman with a first placental tissue
explant; [0048] (c) contacting the explant with a first treatment
and determining the concentration of PP13 of the explant after the
first treatment; [0049] (d) repeating steps (b) and (c) with one or
more additional explants and treatments; and [0050] (e) determining
the difference between the concentrations of PP13 after the
treatments and a corresponding normal level of PP13, the treatment
resulting in the smallest difference being the most effective.
[0051] This aspect of the invention may be referred to at times as
the indirect method.
[0052] In accordance with this aspect of the invention, placental
tissue explants that have been standardized for their response in
the presence of various medications may be used to assess the
displacement/augmentation/blockade effect of various pregnant woman
bodily substances on the effect of the medications on the standard
tissue explants. In a preferred embodiment, the standardized
placenta explants are cryo-preserved before use.
[0053] Also included in this aspect of the invention is a
diagnostic kit for carrying out the method of the invention
comprising (a) a set of anti-preeclampsia drugs; and (b) a set of
standardized placenta explants. In one embodiment, the kit further
comprises computer software providing a calculation model to
determine the effectiveness of the drugs based on the measured PP13
values. Also included in the invention are kits to measure PP13
adjusted to be used in the method of the invention.
[0054] As illustrated in FIG. 1, placenta explants (either standard
ones or ones obtained from the woman) may be grown in culture
conditioned medium for 48 hours or longer in the presence of
various putative anti-preeclampsia medications. In accordance with
the first aspect of the invention in the case of a woman's own
tissue, a comparison is made of PP13 released in conditioning
medium with/without medication. In accordance with the second
aspect of the invention in the case of a standardized tissue from
another source, the tissue is apportioned: one portion is used to
measure the impact of drugs in the absence of the woman's bodily
substance whereas the other portion is tested in their presence. In
the case of a woman at risk for preeclampsia, her bodily substance
influences PP13 release as compared to explants that were not
exposed to the patient's bodily substance. The comparison enables
one to assess the value of the treatment with the medication in
view of molecules included in the bodily substance of the patient.
In all cases, the culture supernatant is collected, centrifuged,
and assayed, for example in an ELISA in-vitro assay, to determine
the level of PP13 released to the medium from the explant. The
level of released PP13, adjusted to the protein level or tissue
weight and culture viability and compared to a control and standard
conditions, is used to assess the impact of the medication.
[0055] The comparative analysis of PP13 release enables one to
identify which medications are capable of bringing PP13 release
back to its normal level as in unaffected patients, and such effect
is taken as an indication of the curative drug effect. Based on the
above, it would be possible to select one of a plurality of various
candidates of medications/combinations of medications of the
currently existing protocols that would have the highest likelihood
of being effective.
[0056] According to the current invention, a woman of established
risk can be followed throughout her pregnancy, and her risk is
assessed in order to verify how various treatments affect her risk.
PP13 from bodily substances is used in this invention to evaluate
the woman's risk. Accordingly, the analysis is not based on a
plurality of cases and controls but is adjusted to individuals and
their specific responses. In this way it is possible to assess
therapeutic benefits/effects and to differentiate between them for
individuals, thereby minimizing the trial and error process.
[0057] The term "abnormal level of PP13" may be defined in relation
to the gestational age according to the three major pregnancy
periods: [0058] (a) Gestational weeks 6-13: high-risk is associated
with low PP13 value (PP13 at the lower population quartile or, in
one embodiment, PP13 multiple of the Median (MoM) below
approximately 0.45). [0059] (b) Gestational weeks 14-25: a steep
increase of PP13 values as compared to the values in the previous
period, or as compared to the values of a normal risk group of
pregnant women. In one embodiment, the increase in PP13 values has
an average slope of 7. [0060] (c) Gestational week 26-to-delivery:
PP13 value above normal (in one embodiment with a MoM of >1.5 of
the highest quartile than or higher than values calculated from a
plurality of normal pregnant women).
[0061] Accordingly, a drug benefit is determined in relation to its
ability to bring the PP13 level of the respective pregnancy period
back to the normal level. Thus, in the first trimester a drug
benefit is determined according to its ability to elevate PP13
level/release to the normal (higher) level. In the second trimester
it is assessed by a drug ability to reduce the steepness of the
slope of change in PP13 release from high (e.g. 7) to normal (e.g.
<3). In the third trimester the benefit is judged according to
the ability to reduce the PP13 level back to normal.
[0062] In the following examples it is demonstrated how measuring
PP13 could benefit in the assessment of elevated risk to develop
preeclampsia and how one might use the PP13 risk assessment tool to
identify a beneficial drug or to tailor drug intervention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] In order to understand the invention and to see how it may
be carried out in practice, certain embodiments will now be
described, by way of non-limiting example only, with reference to
the accompanying drawings, in which:
[0064] FIG. 1 shows the procedure for obtaining placenta explants
from the chorionic villi or the apical membrane of the placenta
syncytiotrophoblast. From left to right, the explants are shown at
transfer (6 hours) and after 36 hr in culture. Cultures are grown
in conditioned medium. After 48 hr in culture, the supernatant is
collected, centrifuged and the pellet is diluted in PBS and checked
for the concentration of PP13 by sandwich ELISA using PP13
standards to calibrate the reaction OD to PP13 concentration. The
PP13 level may be normalized to the gestational age according to
the linear regression of the total points and to the protein level
of the explant;
[0065] FIG. 2 is a graph illustrating longitudinal assessment over
time of PP13 level (pg/ml) measured every 2-4 weeks in blood
samples of 52 women who delivered at term a normal baby, and 5
women who developed severe preeclampsia around term. Delivery time
is indicated by vertical lines. Each curve represents the
anticipated serum level of PP13 for a plurality of normal or
diseased women;
[0066] FIG. 3 is a bar graph which shows division of first
trimester PP13 levels (in pg/ml) of 250 normal women into four
quartiles, and the subsequent designation of 50 women at high risk
for preeclampsia to the various quartiles. According to this
method, a shift of the woman from a lower quartile to a higher one
is an indication of the drug benefit;
[0067] FIG. 4 is a bar graph which shows the PP13 values over three
gestational periods of 1179 normal pregnant women, 20 women at risk
for preeclampsia, 40 normal women who were treated with vitamin E,
and 19 women at risk treated with vitamin E.;
[0068] FIG. 5 is a bar graph showing the rate of false positive
results (i.e. cases where the results based on PP13 measurement
indicate that the woman will develop preeclampsia, but in actuality
she didn't) based on measurement of the PP13 serum level during the
1.sup.st trimester (PP13 MoM), based on the 2.sup.nd trimester
assessment of the slope between two time points (PP13 slope), based
on a combination of the two measurements (Combined) and based on a
combination of the measurements in women with low 1.sup.st
trimester values (Contingent);
[0069] FIG. 6 is a graph showing PP13 (pg/nl) release from
trophoblasts in culture over time (days) obtained from women who
have a normal pregnancy or are at risk for preeclampsia, with or
without treatment with vitamin C and magnesium;
[0070] FIGS. 7-11 are bar graphs which demonstrate median results
.+-.95% confidence intervals of PP13 levels (pg/ml) obtained with
different anti-preeclampsia means (such as 20% oxygen--hyperoxia)
and drugs in standardized placental explants obtained from women at
risk (12 women at risk to develop preeclampsia [grey columns] and 3
women at risk to develop HELLP [dotted columns]) and 16 normal
controls [empty columns] *=p<0.01; **=p<0.001. The tissue of
each woman was divided into 16 different portions, exposed each to
diversified conditions and cultured for 48 hours;
[0071] FIG. 7 illustrates the effect of 6% and 20% oxygen on PP13
release;
[0072] FIG. 8 illustrates the effect of 6% and 20% oxygen, and 0.7
and 1.4 mM Mg on PP13 release;
[0073] FIG. 9 illustrate the effect of 6% (FIG. 9A) and 20% oxygen
(FIG. 9B), 0.7 and 1.4 mM Mg, and vitamins C and E on PP13 release.
M--conditioned medium with 0.7 mM Mg; MC--M+vitamin C;
ME--M+vitamin E; MM--conditioned medium with 1.4 mM Mg;
MMC--MM+vitamin C; MME--MM+vitamin E. Best candidates are indicated
by arrows;
[0074] FIG. 10 illustrate the effect of 6% (FIG. 10A) and 20%
oxygen (FIG. 10B), 0.7 mM Mg, and the anti-coagulents heparin and
aprotinin on PP13 release. M--conditioned medium; MH--M+heparin;
MA--M+aprotinin; MM--conditioned medium with 0.7 mM Mg;
MMH--MM+heparin; MMA--MM+aprotinin. Best candidates are indicated
by arrows;
[0075] FIG. 11 show further results using the conditions presented
in FIG. 10. FIG. 11A--6% oxygen+0.7 mM MgCl.sub.2; FIG. 11B--6%
oxygen+1.4 mM MgCl.sub.2; FIG. 11C--20% oxygen+0.7 mM MgCl.sub.2;
FIG. 11D--20% oxygen+1.4 mM MgCl.sub.2. M--conditioned medium with
0.7 mM Mg; MH--M+heparin; MA--M+aprotinin;
MHA--M+heparin+aprotinin; MM--conditioned medium with 1.4 mM
Mg.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
Methods
[0076] Unless otherwise indicated, in all experiments, PP13 was
measured (blinded to pregnancy outcome) in maternal venous serum by
solid-phase sandwich ELISA assay. The PP13 level was calibrated
according to standard curves prepared from calibrated standards of
recombinant PP13. Concentrations are given in pg PP13/ml serum.
[0077] The various clinical studies were approved by the medical
center Internal Review Ethical Committee, and all women enrolled in
the study provided informed consent to allow the use of a small
volume of their body fluids to determine the level of PP13.
Patients were not randomized for any treatment but the decision to
treat was based on the standard of medical care in the hospital as
best suited to each individual patient. All other details are
described for each example alone.
EXAMPLES
Methods to Assess the Risk for Preeclampsia by PP13 and to Evaluate
its Increase/Decrease
[0078] In the context of assessing the risk for preeclampsia long
before clinical symptoms appear, the following methods in
accordance with the invention may be used to determine the risk
based on PP13 testing: [0079] (1) Measuring a woman's PP13 level by
Sandwich ELISA with PP13 standards and a pair of PP13 specific
monoclonal antibodies as is known, obtaining the data in pg/ml and
comparing the level to the normal median .+-.95% confidence
interval. A cutoff is then established at an optimal
sensitivity/specificity trade-off to determine a PP13 serum level
required to identify women at elevated risk. A significant
difference of a woman's value from the anticipated normal median
establishes the risk. [0080] (2) Calculating the multiple of the
medians (MoM) of the normal plurality after measuring PP13
according to method (1). In this way the median MoM of the normal
PP13 is established. This is normalized to gestation week (GW)
reference medians of normal PP13 values and further adjusted
linearly to maternal weight or body mass index (BMI). [0081] (3) To
standardize raw PP13 values within and across laboratories,
reference medians for normal outcome at each gestational week are
determined by regressing the raw values of at least 40 raw PP13
values per each gestational week over gestational weeks and
extracting from the regression line the reference gestational week
specific median. MoM is then calculated as follows:
[0081] MoM = PP 13 ij Median j ##EQU00001## Where: i=Gestation Week
and j=Subject. The, MoMs are then regressed over BMI categorized
into 4 values of the BMI quartiles and adjusted accordingly. PP13
results are thus standardized. The use of this procedure enables
the combining/comparing data across laboratories. [0082] (4) The
normal plurality PP13 MoM is defined as 1.0. During the first
trimester, MoM=0.45 provides a cutoff under which the patient
likelihood of developing preeclampsia is at least 4 times higher
compared to the normal population with a >80% sensitivity and
>85% specificity. Accuracy is defined by why or receiver
operational characteristic (ROC) curves where an area under the
curve (AUC) of 0.5 provides no prediction, an AUC>0.75 with 95%
confidence interval (CI) of [0.65-0.85] provides a fair prediction
and an AUC>0.8 with 95% CI [0.7-0.9] provides very good
prediction with P<0.05 and above. Women with MoM below 0.45 are
considered as being at elevated risk. The use of MoM is exemplified
in Table 1. During the first trimester, in GW 5-10 and 11-15, women
with elevated risk to develop preeclampsia have a PP13 MoM of 0.14
and 0.17, respectively, which are significantly below the normal
values as indicated by p<0.001. In the second period of
gestational weeks 16-20 and 21-25, the respective MoMs increase
from the earlier very low levels to 0.59 and 1.08 corresponding to
p<0.05 and 0.39, respectively. The example above shows the
benefit of the MoM method in the first trimester and the need to
switch to another method based on PP13 change over time for the
subsequent period as detailed in (5) below. [0083] (5) Measuring
the slope of PP13 change over time by performing two tests of PP13,
a few weeks apart, and calculating PP13
Slope=(PP13.sub.GW2-PP13.sub.GW1)/(GW.sub.2-GW.sub.1), where 1 and
2 represent GW of an earlier and a later time point, respectively,
between which the slope was measured. Comparing to a slope of a
plurality of normal pregnant women which is much lower compared to
women of elevated risk for preeclampsia provides an additional risk
parameter. A cutoff is then established to identify a PP13 Slope
required to identify women at elevated risk at an optimal
sensitivity/specificity trade-off. [0084] (6) Estimating the
woman's likelihood ratio (LR) to develop the pathology based on a
model. In this approach the mode involves estimating the LR for the
possibility of a woman to develop preeclampsia given the PP13 level
relative to the possibility of a normal outcome. Examination of the
MoM values showed that PP13 distribution cannot be fitted to a
Gaussian distribution, either un-transformed or after a log
transformation. Therefore, logistic regression was performed in
order to model LRs, assuming PP13 level determines the risk. The
logistic regression provides the odds ratio (OR) for preeclampsia.
It may then be computed:
[0084] LR.sub.PP13=OR.sub.PP13/P where P is the percentage of
preeclampsia in the pregnant population.
[0085] For any of the methods described above, the data is fit into
a statistical model to plot the receiver operating characteristic
(ROC) curves to evaluate the cutoffs of the measures that are
required to establish the sensitivity and specificity to
distinguish between cases (of preeclampsia) and controls. The use
of MoMs, Slopes and LRs were found to be independent of the
population examined and the lab that performed the testing, whereas
PP13 concentration varied according to the above. Thus, the use of
the three latter measures provides independent population standards
whereas the exact concentration (pg/ml) may vary between population
groups and laboratories.
Longitudinal Monitoring
[0086] FIG. 2 depicts results of longitudinal monitoring over time
of PP13 in serum of normal and preeclamptic pregnant women.
Accordingly, one could identify several parameters to differentiate
normal unaffected women from preeclamptic ones. For monitoring drug
effectiveness, an effective drug should decrease the differences
between normal and high risk patients. Table 1 presents the data of
FIG. 2 after obtaining the MoM values. Statistical analysis was
carried out to compare PP13 MoM normal and preeclamptic values in
each corresponding testing period. The results indicate that during
the 1.sup.st and the 3.sup.rd trimesters but not during the
2.sup.nd trimester Mom PP13 values are significantly different
between preeclamptic and normal women (preeclamptic being very low
in the first trimester and very high in the third while normalizing
in the second). Furthermore, the table shows how treatment with
anti-coagulants as compared to placebo can modify the MoM of
un-treated at-risk women (but not of controls).
TABLE-US-00001 TABLE 1 PP13 MoM throughout Pregnancy Median MoM
(.+-.95% Confidence Interval) Placebo High-Risk for Anti-Coagulants
Normal Preeclampsia Normal High-Risk for Preeclampsia GW Risk (n =
48) (n = 5) (n = 5) (n = 3) 5-10 1.00 0.14** 1.00 0.17** (0.16)
(0.03) (0.16) (0.05) 11-15 1.00 0.17** 1.00 0.48* (0.25) (0.06)
(0.25) (0.12) 16-20 1.00 0.59* 1.00 0.73 (0.29) (0.09) (0.29)
(0.23) 21-25 1.00 1.08 1.00 0.91 (0.18) (0.03) (0.18) (0.23) 26-30
1.00 1.49* 1.00 1.18 (0.26) (0.31) (0.26) (0.25) 31-35 1.00 1.72**
1.00 1.27 (0.12) (0.1) (0.12) (0.27) 36-40 1.00 1.76** 1.00 1.21
(0.14) (0.17) (0.14) (0.21) 40-45 1.00 1.08 1.00 1.00 (0.73) (0.32)
(0.37) (0.34) Outcome All Normal 4 Severe Preeclampsia All 1 Severe
preeclampsia, 1 Mild 1 Normal Normal Preeclampsia 1 Normal (*p <
0.05, **p < 0.001)
1) First Trimester
[0087] First trimester PP13 could serve as a measure to assess the
risk for a later development of preeclampsia. According to FIG. 2
and Table 1, in the first trimester, the majority of the women who
will go on to develop preeclampsia have very low levels of PP13
while most women who will have a normal outcome have higher PP13
values. In one study summarized in Table 2 and FIG. 3, the PP13
level was measured at 8 weeks of gestation from 50 cases who went
on to develop preeclampsia and in 290 cases with normal outcome.
The method of multiples of the gestation-specific median (MoM) was
used, and at MoM cutoffs of 0.45, the false positive rate was 10%
and the sensitivity 87%. This means that 43 out of 50 preeclampsia
cases and 29 out of 290 normal were identified as being at high
risk. Thus, while the risk for PP13 in the population is 5%, in the
group with PP13 below 0.45, the frequency of preeclampsia was 59%
(more than 10 times above the frequency in the population not
tested).
[0088] Sensitivity and specificity were calculated from the
receiver operating characteristic (ROC) analysis based on the
indicated MoM cutoffs of 0.45. Sensitivity value is provided in
Table 2 in percentile for 10% false positive rate. The odds ratio
for developing the pathology was determined by two methods of
calculations:
[0089] 1) modeling, that calculate the Odds ratio as already
described above after taking into consideration the prevalence of
the pathology in the population, and
[0090] 2) the quartile assessment procedure, of calculating of the
odds ratio for the development of preeclampsia based on the
comparison of PP13 in the lowest quartile (25%) versus PP13 in the
third quartile (75th percent).
[0091] The results of these two methods of calculations are
provided in Table 2 and the illustration of the quartile method is
provided in FIG. 3. According to FIG. 3, women who had tested in
the 8 h gestational week were followed until delivery. 290 had
normal delivery. Dividing them into four groups enabled to identify
the four quartiles of PP13 with the respective PP13 concentration
in each quartile being 0-75 pg/ml, 76-139, 140-229, and above 229.
The preeclampsia cases were then assigned to the 4 quartiles
according to their PP13 values. FIG. 3 shows that 86% of all women
who went on to develop preeclampsia were in the lower quartile, 8%
were in the 2.sup.nd quartile, 4% in the 3rd quartile and 2% in the
4.sup.th.
Example 1
Assessment of the Effectiveness of Treatment by Low Dose
Aspirin
[0092] If in the period specified above a medication is used, it is
anticipated that it will bring a woman's PP13 level to the 2nd or
3rd or even the 4th quartile, corresponding to her reduced
likelihood of developing preeclampsia. In the example described in
Table 2, women with elevated risk to develop preeclampsia were
orally treated from GW8 with a dose of 100 mg/kg aspirin ("low dose
aspirin") for either 2 or 3 weeks. It has been suggested that
aspirin given early enough could reduce the risk of later
development of preeclampsia. Accordingly, women who were treated
were anticipated to have lower risk to develop preeclampsia and
their outcome should also be improved.
[0093] In the study shown in Table 2, of 150 women tested as being
at high risk in the 8.sup.th week, 50 were not treated, 50 were
treated with aspirin for two weeks and 50 were treated for 3 weeks.
The results showed that in the untreated group, most women remained
in the low PP13 quartile. In the groups treated for 2 or 3 weeks,
the numbers of patients in the 2.sup.nd and 3.sup.rd quartiles were
elevated significantly compared to the first quatrile. The
calculation of their anticipated risk was reduced accordingly.
Delivery outcome corresponded to the risk assessment, where the
number of preeclampsia cases was significantly lower in the treated
vs. untreated groups.
[0094] Accordingly, the frequency shift from 1.sup.st to 2.sup.nd
and 3.sup.rd quartile could serve as a measure to assess the
reduction in the risk to develop preeclampsia and the effectiveness
of a treatment.
TABLE-US-00002 TABLE 2 Patients Allocated to PP13 Quartiles.
Parameter Untreated 14 days Aspirin 21 days aspirin Sensitivity (%)
87% (80-94) 40 (26-56) 30 (14-46) Frequency of 1.sup.st 43/50 20/50
14/50 quartile cases Frequency of 2.sup.nd 4/50 24/45 30/50
quartile cases Frequency of 3.sup.rd 2/50 5/50 5/50 quartile cases
Odds ratio (by 77.6 5.9 3.3 modeling) Odds ratio (by 73.7 5.6 3.2
quartiles) Outcome All 50 24 Preeclampsia 18 Preeclampsia
Preeclampsia 26 Normal 33 Normal
[0095] Sensitivity in percentile values are shown when the
specificity was fixed to 90% (95% CI: 86%-93%), corresponding to
having 29/292 cases of false positives (10%).
Example 2
Assessment of the Effectiveness of Treatment by Anti-Coagulant
Drugs
[0096] Women were identified as being at elevated risk and were
treated from the 8.sup.th week of gestation with anti-coagulants
(low molecular weight heparin, aprotinin or others) given daily for
2 weeks. Their PP13 MoM was found to be elevated to 0.48 MoM
(GW11-15) (P<0.05), and 0.73 (GW16-20), respectively, with the
latter being practically indistinguishable from the normal level
(1.+-.0.29, Median normal MoM.+-.95% Confidence Interval). PP13 MoM
of women with normal risk was not affected. The corresponding
outcome of the treated women was: with no treatment, all 5 women
with elevated risk developed severe preeclampsia around term,
whereas in the treated group one developed severe preeclampsia, one
mild preeclampsia and one was unaffected.
Example 3
Assessment of Drug Benefit Using Placental Extract
[0097] An alternate method of assessing drug benefit is by using
placenta villi (cells or explants) obtained during gestation week
9-10 from pregnant women undergoing chorionic villi sampling. The
placenta cells/explants were cultured for 48 hr and PP13 was
measured in the culture medium by ELISA (in the same manner as
described in FIG. 1). The results are shown in Table 3 below.
[0098] Table 3 shows that for the 3 cases of preeclampsia (cases
#3, 4 and 5), the amount released under 6% oxygen (normoxia) is
much lower (3,010, 3,500 and 6,300) as compared to 14,100 and
15,700 in normal women (cases #1 and 2). After 48 hours incubation
with the anti-oxidant vitamin C, that has shown promise in treating
high-risk women, the level of PP13 release is brought up almost to
the normal level in all 3 high risk women, reaching 12,030, 9,230,
and 15,790, respectively (i.e. 3-4 times higher). Under 20% oxygen
(hyperoxia), PP13 release increased to 5000, 4,300 and 7,900,
respectively, due to the oxygen itself (approximately by a factor
of 2 as compared to 6% oxygen: 3,010, 3,500 and 6,300 pg/ml), while
there is no additional effect of vitamin C.
[0099] As can be seen, not all individual women treated this way
show the same effect, indicating the potential of the method of the
invention to be further used to consider discontinuing the
treatment, elevating the drug level or selecting a different
treatment for individual patients. Although the use of a placental
extract for assessing preeclampsia risk saves a woman from exposure
to an un-necessary medication, she is exposed to an interventional
procedure of risk. Thus, this is suitable only to those women
undergoing an interventional sampling of placenta tissue in any
event.
TABLE-US-00003 TABLE 3 PP13 Release from Cultured Chorionic villi
of GW 9-10 PP13 Pregnancy Case # Risk Oxygen (%) Vitamin C (pg/ml)
outcome 1 Normal 6 No 14,100 Normal Yes 14,300 20 No 14,250 Yes
14990 2 Normal 6 No 15,790 Normal Yes 15,390 20 No 15,500 Yes
15,300 3 High-Risk 6 No 3,010 Preeclampsia Yes 12,030 20 No 5,000
Yes 5,100 4 High-Risk 6 No 3,500 Preeclampsia Yes 9,230 20 No 4,300
Yes 4,421 5 High-Risk 6 No 6,300 Preeclampsia Yes 15760 20 No 7,900
Yes 8,100
2) 1.sup.st-to 2.sup.nd Trimester Slope
Example 4
Assessment of Drug Benefit Using the 1.sup.st-to 2.sup.nd trimester
slope
[0100] As already demonstrated in FIG. 2, in normal women PP13
level is only moderately changed between the 1.sup.st and the
2.sup.nd trimester. The slope may be calculated as follows:
Slope=(PP13.sub.(2nd trimester)-PP13.sub.(1st
trimester)/GW.sub.(2nd trimester)-GW.sub.(1st trimester)).
Normal vs. preeclampsia slopes are shown in FIG. 4, and the cutoff
between normal and preeclampsia to reach 80% sensitivity is a slope
of 3.5. The slope helps to further verify the risk for
preeclampsia. The results in FIG. 4 show that in the cases of
preeclampsia at 6-10 weeks, the PP13 level is lower than in the
normal cases, and early application of vitamin E doubles PP13
release towards the normal level without affecting PP13 release in
normal patients. At 16-20 weeks, no significant effect of vitamin E
can be demonstrated. At 24-28 weeks, when PP13 release in
preeclamptic women is higher than in normal women, vitamin E
reduces PP13 release back to the normal level.
[0101] FIG. 5 shows how the level of false positives for a fixed
prediction sensitivity is reduced by a combined analysis using both
PP13 MoM level in the 1.sup.st trimester and the
1.sup.st-to-2.sup.nd trimester slope. The figure indicates that
either first trimester MoM and the slope provide each a 15% false
positive rate with 80% sensitivity. Taking the two parameters
combined--both first trimester MoM and first-to-second trimester
slope by way of combined analysis enabled reducing the false
positive rate to 6% without losing sensitivity. In a contingency
approach ("Contingent"), only women with low first trimester MoM
were tested again and the combined analysis shows that for the same
sensitivity, the false positive rate is 8%. Accordingly, it appears
that second trimester testing is a must for those identified at
risk in the first test.
[0102] If after establishing the risk by two tests the women at
high risk are treated daily by administering the anti-oxidant
vitamin E, the treated women have a lower slope (FIG. 4). From the
relatively large confidence level one can see that not all women
were affected in the same way. Thus, the approach can be further
used to enable one to see how the slope can be reduced by
medications and its correlation to the reduced risk to develop the
pathology, thereby considering discontinuing the treatment,
elevating the drug level or selecting a different treatment for
individual patients. The combined and contingent approaches
indicate that multiple testing is necessary only for women at risk
whereas low risk patients may avoid repeated testing.
Example 5
Assessment of Drug Benefit Using Trophoblasts
[0103] The subject can also be monitored by looking at trophoblasts
obtained from amniotic fluid. For example, the trophoblasts may be
grown for two weeks under culture conditions with and without a
combination of 1.4 mM MgCl.sub.2 and Vitamin C. Trophoblasts in
culture from women who went on to develop preeclampsia showed a day
to day elevated PP13 release (indicating elevated risk to
preeclampsia) as compared to trophoblasts obtained from normal
women, whose PP13 release remained practically unchanged (FIG. 6).
Culturing the trophoblasts with Mg and Vitamin C had no effect on
the normal cultures but prevented the increased PP13 release
compared to untreated trophoblasts, indicating a method for
determining the treatment effectiveness/ineffectiveness for
individual cases. Again, it is anticipated that there will be cases
that will not respond, and thus the procedure could be used to
"tailor" treatment by continuing, discontinuing or replacing with a
different drug.
3) Third Trimester
Example 6
Assessment of Oxygen Benefit
[0104] The following results were obtained with placental (villous)
explants from 16 cases of normal women, 12 cases of preeclampsia
and 3 cases of HELLP women, which were cultured in DMEM/F12 for 48
hours at 6% or 20% O.sub.2. Conditioned media was collected after
culture and tested for total protein and PP13. PP13 release was
related to total protein.
[0105] As can be seen in FIG. 7, in cultures grown under an
elevated (20%) oxygen level, oxygen had no effect on PP13 release
from placental extracts obtained from normal women but decreased
significantly the release of PP13 from placental extracts obtained
from preeclampsia women, and even more so in placental extracts
obtained from HELLP cases, indicating that the oxygen was harming
the placental tissue.
Example 7
Assessment of Benefit by Combination of Oxygen and Mg
[0106] The explants tested in Example 6 were retested using, in
addition to the two oxygen levels, two different levels of
magnesium, 0.7 mM and 1.4 mM.
[0107] As can be seen in FIG. 8, in the culture grown at elevated
oxygen (20%) and MgCl.sub.2 (1.4 mM)--the explants obtained from
normal pregnant women were not affected but the preeclamptic
explants were brought back to the normal level by magnesium in 20%
but not in 6% oxygen. The level of PP13 release in HELLP cases was
reduced by 20% O.sub.2 to below the normal level.
Example 8
Assessment of Benefit by Combination of Oxygen, Mg and Vitamins C
& E
[0108] The experiments described in Examples 6 and 7 above were
repeated with the addition of Vitamins C & E. As can be seen in
FIG. 9, the addition of the vitamins helped to bring PP13 back to
the normal level in 6% oxygen, particularly at elevated magnesium.
Note that the latter combination also affected the HELLP cases.
Example 9
Assessment of Benefit by Combination of Oxygen, Mg and
Anticoagulents
[0109] The experiments described in Examples 6 and 7 above were
repeated with the addition of various anti-coagulants. As can be
seen in FIG. 10, the addition of the anti-coagulants was beneficial
particularly if they are combined with magnesium.
Example 10
Assessment of Benefit by Combination of Oxygen, Mg and
Anti-Coagulants
[0110] In this example, the effect of heparin and aprotinin on the
PP13 release from villous explants of normal, preeclamptic and
HELLP placentas was tested in vitro under the conditions of the
previous examples. The results are presented in FIGS. 11A-11D.
[0111] In FIG. 11A, the explants were cultured under normal
conditions (normoxia (6%) and normal Mg (0.7 mM)). Significantly
more PP13 was released from explants derived from preeclampsia and
HELLP patients than from the normal controls. In FIG. 1B, culture
under normoxia and elevated Mg (1.4 mM) resulted in elevated
release of PP13 in the HELLP patients. In FIG. 11C (hyperoxia (20%)
and normal Mg), significantly less PP13 release occurred from
explants of preeclampsia as compared to normoxia, while the control
remained at the same level. An almost complete halt in PP13 release
from the HELLP patients was noted. An increase in Mg (FIG. 1D)
brought about an elevated release of PP13 in the HELLP
patients.
[0112] With respect to the anti-coagulants, under normoxia and high
Mg (FIG. 11B), aprotinin brings PP13 release in explants derived
from HELLP patients almost back to normal. Under hyperoxia and
normal Mg (FIG. 11C), heparin brings PP13 almost back to
normal.
[0113] Thus, the method of the invention may be used to forecast
which combination of treatments will be the most effective in
overcoming the risk for developing preeclampsia.
[0114] It is very important to note that in all of the examples
described above, a severe subtype of preeclampsia--HELLP--is not
affected in the same way as preeclampsia. Thus, the explant system
could help in selecting a proper treatment by testing the effect in
vitro using the explant system and assessing the proper treatment
for the individual woman and disease. Since explants can be stored
by cryopreservation, it is also possible to standardize them for
further evaluation and tailoring of drug intervention.
Example 11
Assessment of Benefit by Administering VEGF
[0115] sflt1, a soluble form of the vascular EGF (VEGF) receptor,
was found to be at a higher serum level in the third trimester in
women who went on to develop preeclampsia 5 weeks later. This
molecule competes with the native blood cell receptor for the
hormone VEGF. Experimental models have shown that administering
VEGF could prevent/reduce the severity of preeclampsia. One way to
follow in-vitro the benefit of the treatment is to measure PP13
level and, if decreased back to the normal level, it could serve to
assess the benefit of the treatment.
4) Longitudinal Monitoring
Example 12
[0116] The following is a prophetic example describing how the
method of the invention may be used to follow the risk of
preeclampsia of a woman throughout her pregnancy.
[0117] The MoM of PP13 level is defined as "1" for a plurality of
unaffected (normal) women. If the maternal serum is tested at 10
weeks (1st trimester) and a woman's PP13 level corresponds to 0.11
MoM as defined by a statistical plurality of pregnant women at the
respective gestational week, the woman is established as being at
high risk for preeclampsia. PP13 MoM=0.45 is the cutoff of 80%
specificity and 85% sensitivity. From the model, her likelihood
ratio (LR) is found to be 9 times above normal (5%) or at a risk of
45%. If after two weeks of treatment the MoM doesn't cross the
cutoff--she remains at high risk.
[0118] Then, 4 weeks later (already 2.sup.nd trimester, GW=16), she
is re-tested for PP13. Her MoM is now 1.24, and the calculated
slope between the two points is 7 while the slope cutoff is 3.0.
Thus, the woman is defined as being at continued high risk. From
the model, her LR is calculated to be 8.4 times above normal
(risk=42%). Her average risk is thus 43.5%. Another treatment with
anti-oxidants is now evaluated and her MoM returns to be 1.0,
indicating her risk has now been reduced. From the model her LR=2
(corresponding to 10% risk).
[0119] She is then tested a third time at 30 weeks (3.sup.rd
trimester) and her MoM is found to be 1.5, whereas the high-risk
cutoff of that week is 1.4 MoM. Accordingly, her LR=8.5 times above
normal, and her risk is thus again 42.5%. She is now treated again
with anti-oxidants and re-tested at 34 weeks. Her MoM is then found
to have declined to 1.2 (below cutoff). Her LR=2, risk is 10%. The
treatment is now continued and she delivers at term with blood
pressure 85/135 and proteinuria 1+ (not considered as
preeclampsia).
Example 13
Standardization of Explants to Measure the Effect of Various Drugs
(Prophetic Example)
[0120] A woman was identified to be at risk for preeclampsia by a
first trimester marker such as PP13 or PP13 combined with
Doppler--how might explants help to tailor a preventive treatment
to her?
[0121] Scenario 1: Simple Direct Tailoring with a Diversity of
Drugs. [0122] We have standardized culture conditions for first
trimester explants or explants obtained after delivery. [0123]
Explants could be the ones made of patients after delivery or those
obtained by Chorionic villi sampling (CVS) at gestational weeks
10-12 or other placenta biopsies as the case may be. [0124]
Standardization means defined median viability index, protein
content in the explant, total protein released to the medium, PP13
content in the explant and PP13 release from the explant, among
others. [0125] Drug effect in-vitro is the effect of drug applied
to the culture medium on PP13 release from the placenta explant
after 24 hr to 7 days. The effect is compared to the baseline
release as measured without the drug. [0126] The Therapeutic Index
is thus the in-vitro relative effectiveness of the drug when
applied to explants as measured by the difference between PP13
release with (PP13.sub.D1) and without (PP13.sub.0) the drug by the
equation: (PP13.sub.0-PP13.sub.D1)/PP13.sub.0, after normalizing to
viability, protein content etc as described above, given all other
parameters are the same. [0127] It is important to note that in the
first trimester (gestational weeks 6-13) PP13 in diseased patient
is lower than Normal. Thus the therapeutic index is to return PP13
release is calculated as drugs that elevate PP13 release. In the
third trimester (gestational week 26 and above), PP13 is higher in
preeclamptic patients than normal. Thus the therapeutic index is
decreasing PP13 release back to Normal. [0128] According to the
in-vitro therapeutic index one drug or a drug combinations are
selected for the in-vivo interventional medication treatment.
[0129] Follow up: after selecting the drug to be administrating to
the patient, bi-weekly blood testing follow up is recommended to be
carried out by measuring the PP13 in the blood and calculating
[0130] PP13 slope=(PP13GW2-PP13.sub.GW1)/(GW.sub.2-GW.sub.1), where
GW.sub.1 and GW.sub.2 represent gestational week at the first and
second period of PP13 testing, respectively. The result of the
formula defined as the slope that was calculated for each
individual. This one is compared to the typical median slope for
the cases of preeclampsia vs. normal cases. If for the first period
(gestational week 6-13) the normal slope is 3.1 and the
preeclamptic slope is 10 and after treatment the slope is going
down, every test compared to the one before, than it indicates that
the treatment is effective. Otherwise--it is recommended to switch
to the drug with the second best therapeutic index.
[0131] Scenario 2: Tailor to Preeclampsia "Types" [0132] We
measured in the body fluid of a woman a very low level of PP13 in
the first trimester, indicating that she is at elevated risk for
preeclampsia. [0133] We verify the various other features (like low
Doppler pulsatility Index or low PAPP-A). [0134] Based on this
set--we type the patient to a preeclampsia group A that corresponds
to one type of preeclampsia (such as early onset preeclampsia).
Another set of patients will have only low PP13 in the first
trimester but none of the other and is referred to as Group B.
[0135] In-Vitro explants of Type A are subsequently found to be
affected by Drug 1 whereas the explants of Group B the Therapeutic
Index indicates that only Drug 2 is effective. [0136] Accordingly,
once the markers set of a patient is identified to be belonging to
group A, Drug 1 will be selected for treating group 1 and vice
versa. [0137] Follow up: after selecting the drug to be
administrating to the patient, bi-weekly blood testing of PP13 is
carried out for the determination of the slope as described
above.
[0138] Comment--From what we know today on preeclampsia diversity,
the approach could at least narrow down significantly the list of
suitable therapeutics means to 1-2 candidates for a group. This
seems to be most suitable for cases of IVF where many tests are
carried out for each woman. Thus a large set of markers could be
used to fine-tune the "patient type (group).
[0139] Scenario 3: Indirect method [0140] We standardized growth
conditions for placenta explants obtained from after delivery from
patient A of known outcome. [0141] We find the drug effectiveness
on these sets of explants. [0142] After testing many drugs a drug
effectiveness scale is developed. [0143] We expose these
standardized explants with their scaled drug effectiveness index to
the respective drugs in the presence of serum from normal vs. serum
from a patient at elevated risk for preeclampsia (Table 1). [0144]
We found the most effective drug in the setting of exposure to
drugs in the setting of incubation with normal patient serum and
the compared effect in the presence of serum from woman identified
to eb at elevated risk for preeclampsia. [0145] We choose the drugs
that is the less impaired by the patient serum and apply this drug
onto the patient in-vivo. [0146] that is the lesser to be impaired
by the serum of the affected patient and tailor it to this patient.
[0147] This scenario assume that at elevated risk for preeclampsia,
the patient body fluids contain various factors such as sflit,
estriol, shbg or others that impair/enhance drug effectiveness and
presumably even causing preeclampsia.
[0148] If so, drugs that appears beneficial to standardized culture
explants wouldn't work when applied together with the patient
samples. Results of an actual experiment are presented in Table 4
below. It can be seen that the serum from the patient at high risk
for preeclampsia caused serious inhibition of many of the types of
treatment. This method allows the selection of the treatment most
likely to prove effective.
TABLE-US-00004 TABLE 4 Blocking impact of patient serum on the
Therapeutic Effectiveness of anti-preeclamptic drugs as assessed in
placenta explants by measuring the release of PP13 to the culture
medium Therapeutic Effectiveness of anti-PP13 medications on PP13
release from placenta explants when applied with serum from normal
and preeclamptic patient Serum from a Normal Serum from a Patient
at Treatment Patient high risk for preeclampsia Aspirin 74% 40%
Heparin 56% 20% Aspirin + Heparin 85% 67% Aspirin + Heparin + O2
97% 85% Vitamin E 30% 0% Magnesium 50% 20% Mg + Vitamin E 95%
80%
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